In-situ quantitation of genome release of Japanese encephalitis viruses by quantum dot-based single-virus tracking

•A strategy for dual-labeling of viral envelope and genome with QDs and multiple molecular beacons (MMBs) was developed.•The combination of SVT and the in-situ pH quantification of endosomal acidification during viral infection was achieved.•Our results showed that the specific virus-endosome fusion events of JEV predominantly occurred in the maturing endosomes.•We addressed the question that where and how the viruses harness endosomal compartments and release their genome.